Evaluation of phytochemical components, cytotoxicity, and molecular docking of Anacyclus pyrethrum and Commiphora myrrha for formulating a herbal topical anaesthetic gel development

Abstract

Background

Synthetic intraoral topical anesthetics, such as lignocaine and benzocaine can cause adverse effects in pediatric dentistry, creating a need for safer plant-based alternatives. Anacyclus pyrethrum (A. pyrethrum) and Commiphora myrrha (C. myrrha) have traditionally demonstrated anesthetic and analgesic properties. Therefore, we aimed to evaluate the phytochemical composition, cytotoxicity, and molecular docking interactions of A. pyrethrum and C. myrrha extracts for potential use as novel herbal topical anesthetic gels.

Methods

Ethanolic extracts of A. pyrethrum roots and C. myrrha resin were prepared by Soxhlet extraction and maceration, respectively. Phytochemical profiling was performed using High-Performance Liquid Chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and Fourier-Transform Infrared Spectroscopy (FTIR). Cytotoxicity was assessed in L929 fibroblasts and neural cells using the MTT assay. Molecular docking and codocking of pyrethrin and furanoeudesma-1,3-diene with neuronal sodium, potassium, and GABA-A receptors were performed.

Results

HPLC and GC-MS confirmed the presence of bioactive compounds, including pyrethrin and furanoeudesma-1,3-diene, and the functional groups were validated by FTIR. Cytotoxicity assays revealed high biocompatibility, with half-maximal Inhibitory Concentration (IC₅₀) values of 53 ± 0.21 µg/mL for A. pyrethrum and 54 µg/mL for C. myrrha. Molecular Docking studies showed strong binding affinities (−6.1 to −8.2 kcal/mol) across target receptors, and co-docking demonstrated synergistic ligand–receptor interactions.

Conclusion

Phytochemical, cytotoxic, and molecular docking analyses confirmed the anesthetic potentials of A. pyrethrum and C. myrrha. These findings support their further development as safe plant-based topical anesthetic gels.

INTRODUCTION

Pain is an unpleasant sensory and psychological experience from actual or potential tissue damage, commonly associated with dental treatment [1]. Pain and discomfort influence children’s behaviors and anxiety during dental visits [2]. Inadequate pain management can lead to negative reactions and fear, making it challenging for dentists to foster a positive outlook. Therefore, treat young patients with minimal discomfort and pain has become a primary goal of pediatric dentists [3].

The word anesthesia was derived from the Greek words ana ("without") and aesthesis ("feeling") [4]. Local anesthesia is the loss of sensation in a circumscribed area due to depressed nerve excitation. It can be applied as a topical anesthetic, for infiltration, or as a nerve block. Topical anesthesia, or surface anesthesia, is effective only 2–3 mm from the mucosal surface. It produces superficial loss of sensation through direct application of solutions, ointments, gels, or sprays, primarily to reduce pain from needle insertion for local anaesthesia administration [5].

Common topical agents, such as lignocaine and benzocaine, show increasing adverse reactions, particularly in infants [6]. Lidocaine application often cause an unpleasant taste [7], while benzocaine overdose can cause methemoglobinemia [8]. Consequently, interest in alternative medicines is growing. The World Health Organization reported that 80% of individuals in developing nations primarily depend on herbal and Ayurvedic treatments for basic healthcare [9].

Various plants, including clove (Syzygium aromaticum), neem (Azadirachta indica), turmeric (Curcuma longa), lavender oil (Lavandula spp.), and betel leaves (Piper betel), demonstrated anesthetic and analgesic properties [10]. Youssef ER et al. found clove oil gel produced significantly lower pain scores than lignocaine [11]. Similarly, Havale et al. showed betel leaves and cloves effectively reduced pain and could serve as alternative topical anesthetic agents [12].

Anacyclus pyrethrum (A. pyrethrum; Akarkara) is an endangered herb cultivated commercially for pyrethrin extraction from its roots. Highly regarded in Ayurvedic medicine for antioxidant and antibacterial properties, it contains alkaloids, tannins, triterpenes, flavonoids, sterols, trace metals and phenolic compounds. Its roots contain abundance esters called pyrethrine and N-alkyl amides (pellitorine), enhancing therapeutic properties [13]. Research showed that A. pyrethrum extracts possess anesthetic properties outperforming lignocaine and reduce (needle–stick) pain when combined with Spilanthes acmella [14].

This resin, known as myrrha, obtained from genus Commiphora, has a long medical history. Bioactive components, including steroids, terpenoids, flavonoids, lignans, and sugars, have been identified in C. myrrha. Crude extracts and isolated compounds demonstrated several biological activities, such as anti-inflammatory, antimicrobial, antiproliferative, cardiovascular, and hepatoprotective effects [15]. These extracts show 50% procaine anaesthetic potency [16].

The use of herbal alternatives for topical anesthesia in dentistry remains limited and largely unexplored. While A. pyrethrum is cited for strong local anesthetic action through alkylamides, C. myrrha offers strong analgesic and anti-inflammatory properties. This dual-action approach aims to achieve superior topical anesthesia while managing post-procedural inflammation, a crucial factor in pediatric dental comfort. No studies have evaluated the combined effects of A. pyrethrum and C. myrrha as anesthetic agents, and no detailed pharmacological evaluation has been conducted. Therefore, this study aimed to develop a novel polyherbal topical anesthetic gel combining these plants. The first step was comprehensive phytochemical analysis, cytotoxic assessment, and molecular docking investigation of the selected species.

METHODS

This study was approved by the Institutional Review Board (IRB) of the institution, ensuring ethical compliance with research involving bioactive plant extracts and cell cultures. The study period is from June 2024 to July 2025. The first two stages, drug discovery and development and preclinical research for drug development and evaluation, were conducted in accordance with the guidelines of the Central Drugs Standard Control Organisation (CDSCO). Figure 1 illustrates a flowchart of the study protocol.

Fig. 1. Illustrates the flowchart of the study protocol. A. pyrethrum, Anacyclus pyrethrum; C. myrrha, Commiphora myrrha; FTIR, Fourier-transform infrared spectroscopy; GC-MS, gas chromatography-mass spectrometry; HPLC, high-performance liquid chromatography; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IRB, Institutional Review Board.

1. Extraction of phytochemical constituents

Dried roots of A. pyrethrum were procured commercially from Indianjadibooti (Uttar Pradesh, India; Batch No: Lot-6). C. myrrha resin was obtained from NatureLoc (Kerala, India; SKU: 60810, UPC: A25). The dried roots and resin were ground into a coarse powder using a laboratory mill and passed through a 40-mesh sieve to ensure uniformity. The resultant powder was stored in an airtight container.

Soxhlet and Maceration extraction methods were used to extract bioactive compounds from A. pyrethrum and C. myrrha, respectively. In the Soxhlet extraction method, 2 g of powdered plant material was placed in a cellulose thimble and 250 mL of ethanol, acetone, and methanol were heated to their boiling points for six cycles (4–6 h). The final extract was concentrated under reduced pressure and stored at 4℃ [17]. For maceration, 5 g of powdered C. myrrha resin was soaked in 250 mL of hexane and 250 mL of ethanol in a sealed 250 mL amber flask, agitated at 100 rpm for 48 h at ambient temperature, and then filtered using Whatman No.1 paper. The extract was evaporated under reduced pressure and stored at 4℃ [18].

2. Identification of the active component

The ethanol extracts obtained from A. pyrethrum and C. myrrha were analyzed using High-Performance Liquid Chromatography (HPLC), Gas Chromatography-Mass Spectrometry (GC-MS), and Fourier-Transform Infrared Spectroscopy (FTIR).

The HPLC analysis was performed using an Agilent 1200 Series HPLC system (Agilent Solutions, Santa Clara, California, USA) equipped with a C18 reverse-phase column. The mobile phase used was acetonitrile: water (95:5) for A. pyrethrum [19] and (80:20) for C. myrrha [20] with detection wavelengths of 210 and 240 nm, respectively. Samples were prepared at a concentration of 20 ng/µL in HPLC-grade acetonitrile and filtered through a 0.22 µm syringe filter. A 20 µL sample was injected into the system, and separation occurred under a constant flow rate of 0.8 mL/min. The chromatograms were monitored and the retention times of the peaks were compared with those of the library.

GC-MS was used to separate and identify volatile and semi-volatile compounds in the plant extracts. Ethanolic extracts of A. pyrethrum and C. myrrha were dissolved in Dimethyl sulfoxide (DMSO) at 1 mg/mL, filtered through a 0.22 µm membrane, and 1 µL was injected into a Triple Quadrupole GC-MS/MS system (CH-GCMSMS-02-Labchrom Scientific LLP, Mumbai, India) equipped with a VOC analyzer column. Helium was used as the carrier gas. The oven temperature was initially held at 60℃ for 15 minutes, then ramped at 3℃/min to 280 ℃, with a total run time of approximately 30 minutes. The compounds were identified by spectral matching using the National Institute of Standards and Technology (NIST) and Wiley databases [21].

FTIR spectroscopy was used to identify the functional groups in the plant extracts. The FTIR analysis was conducted using a PerkinElmer Spectrum Two FTIR spectrometer (PerkinElmer, Revvity, Inc., Massachusetts, US). The dried ethanol extracts of A. pyrethrum and C. myrrha were directly applied to Attenuated Total Reflection (ATR) crystals. The scan range was set from 4000 cm⁻¹ to 400 cm⁻¹ with a resolution of 4 cm⁻¹. The spectra were recorded and analyzed to identify the functional groups based on the absorption peaks [21].

3. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay

The MTT assay was performed to assess cell viability and cytotoxic potential of the plant extracts. Biocompatibility was assessed using L929 fibroblasts and a neural cell line. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin, maintained at 37℃ with 5% CO₂. The procedure followed the ISO 10993-5 and ISO 10993-12 guidelines. Cells were seeded at 1 × 10⁴ cells/well in 96-well plates and allowed to adhere overnight. The next day, they were treated with different concentrations (12.5, 25, 50, 100, and 250 µg/mL) of each plant extract dissolved in Dimethyl sulfoxide (DMSO). The final DMSO concentration did not exceed 1 wt. %. After 24 hours of exposure, 10 µL of MTT reagent (5 mg/mL) was added to each well and incubated for 4 hours. After incubation, the media were removed, and the formazan crystals were solubilized using 100 µL of DMSO. The absorbance was read at 570 nm using a microplate reader [22].

4. In-silico molecular docking

Molecular docking was conducted to determine the interactions of key bioactive compounds identified in both A. pyrethrum and C. myrrha with pain-related receptor proteins. In addition to individual molecular docking, co-docking was performed to simulate the combined binding of pyrethrin and furqnoeudesma-1,3-diene. The docking procedures involved preparation of ligands and target proteins.

5. Preparation of Ligand:

The phytocompound pyrethrin (CID: 5281555) from A. pyrethrum and furanoeudesma-1,3-diene (CID: 13874240) from C. myrrha were downloaded from the PubChem database in 3D SDF format. The structures were visualised using BIOVIA Discovery Studio software (Dassault Systems, Vélizy-Villacoublay, France) and converted from a Structure Data File (SDF) format to Protein Data Bank (PDB) format. The ligand was prepared using AutoDock 4.2.6 software (Scripps Research, San Diego, California, US) [23].

6. Preparation of target proteins:

The selected target proteins included voltage-gated sodium channels (PDB ID: 6MVX, 6NAQ, 5EK0, 6AGF, and 7WE4), potassium channels (PDB ID: 3UKM and 6RV3), and Gamma-Aminobutyric Acid (GABA-A) (PDB ID: 6CDU and 6D1S) receptors. Voltage-gated sodium channel (Na_v) subtypes were chosen because of their critical role in the initiation and conduction of pain signals in dental tissues, as well as their high expression in oral mucosal nociceptors (Na_v1.7; PDB: 6MVX) and Na_v1.8 (PDB: 6NAQ). To assess any possible central or peripheral alteration of inhibitory signalling related to topical anesthetic effects, GABA-A receptors were also included.They were determined by the X-ray diffraction method with a resolution of 3 Å and having a single chain named A. The PDB format of the target protein was retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB) PDB database and visualized and optimized using BIOVIA Discovery Studio software. Target proteins were optimized by deleting heteroatoms and water molecules. Polar hydrogen was added and the active sites were determined. Target proteins were converted from PDB to PDBQT format using AutoDock 4.2.6 software. The optimized target protein was subjected to molecular docking and codocking studies using AutoDock [23].

Molecular dynamics simulation

Molecular Dynamics (MD) simulations were performed to evaluate the interaction and stability of the selected phytoconstituents with the target protein. The PDB format of the target protein was retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB) PDB database and prepared by removing heteroatoms and adding hydrogen atoms. The ligand structures were prepared and parameterized using the CHARMM36 all-atom force field [24]. Protein-ligand complexes were subjected to MD simulations using GROMACS (version 2020.1) under physiological conditions [25]. Structural stability and interaction dynamics were determined using Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of Gyration (RG), hydrogen bond analysis, and solvent-accessible surface area (SASA) [26].

RESULTS

1. High-performance liquid chromatography (HPLC)

The chromatograms revealed multiple peaks for both extracts, suggesting the presence of various bioactive compounds. In A. pyrethrum, several prominent peaks corresponding to different phytochemicals were observed, including alkylamides, pyrethrin (RT: 2.53 min), spilanthol (RT: 3.26 min), and phenolic acids (RT: 4.23 min) (Fig. 2A). In C. myrrha, distinct peaks indicated the presence of terpenoids (RT: 3.9 min), sesquiterpenes (RT: 4.4 min), and resin acids (15.7 min) (Fig. 2B). These compounds are known for their therapeutic and esthetic properties. These results confirm that ethanol is an effective solvent for extracting a wide range of bioactive components, supporting its potential use in the development of topical anesthetic gel formulations from these plant extracts.

Fig. 2. High-performance liquid chromatography (HPLC) of the ethanol extract of: A) A. pyrethrum; and B) C. myrrha.

2. Gas chromatography–mass spectrometry (GC-MS)

The A. pyrethrum extract showed prominent peaks corresponding to pyrethrin II, an alkylamide compound with strong anesthetic and neuromodulatory properties. In C. myrrha, the major compound identified is furanoeudesma-1,3-diene (RT: 21.66) known as the major compound in C. myrrha. The corresponding chromatographic peaks and their chemical structures obtained directly from the GC-MS report are shown in Figures 3A and 3B, respectively. The compounds obtained from the ethanolic extracts of A. pyrethrum and C. myyrha has been summarised in Table 1.

Fig. 3A. Gas chromatography and mass spectrometry (GC-MS) of the ethanol extract of Anacyclus pyrethrum.

Fig. 3B. Gas chromatography and mass spectrometry (GC-MS) of the ethanol extract of Commiphora myrrha.

Table 1. Key phytochemical compounds identified in ethanolic extracts via GC-MS.

Plant extract	Retention time (min)	Compound name	Chemical formula	Molecular weight (g/mol)
A. pyrethrum	25.29	2,4-Decadienamide, N-isobutyl-, (E,E)- (Pellitorine)	C14H25NO	223.36
	27.70	(2E,4E)-N-Isobutyldodeca-2,4-dienamide	C16H29NO	251.41
	29.73	(2E,4E)-N-Isobutyltetradeca-2,4-dienamide	C18H33NO	279.46
	25.50	n-Hexadecanoic acid	C16H32O2	256.43
	27.53	9,12-Octadecadienoic acid (Z,Z)-	C18H32O2	280.45
	32.4	Pyretrin II	C22H28O5	372.5
C. myrrha	21.66	3,5,8a-Trimethyl-4,4a,8a,9-tetrahydronaphtho[2,3-b]furan (Furanoeudesma-1,3-diene analog)	C15H18O	214.31
	19.66	Benzofuran, 6-ethenyl-4,5,6,7-tetrahydro-3,6-dimethyl-5-isopropenyl-, trans-	C15H20O	216.32
	24.74	4,4′-Dimethyl-2,2′-dimethylenebicyclohexyl-3,3′-diene	C16H22	214.35
	22.83	(R,5E,9E)-8-Methoxy-3,6,10-trimethyl-4,7,8,11-tetrahydrocycl odeca[b]furan	C16H22O2	246.35
	26.22	Gazaniolide	C15H18O2	230.30

A. pyrethrum, Anacyclus pyrethrum; C. myrrha, Commiphora myrrha; GC-MS, gas chromatography-mass spectrometry.

3. Fourier-transform infrared spectroscopy (FTIR)

Pyrethrins are esters of chrysanthemic acid containing cyclopentenone rings and olefinic chains. Characteristic IR bands included a strong ester carbonyl (C = O) stretch around 1730–1750 cm⁻¹, a conjugated C = C stretch between 1630 and 1690 cm⁻¹, and aliphatic C–H stretches in the 2850–2960 cm⁻¹ region. In one reference synthesis, a pyrethrolone precursor exhibited IR peaks at 1735 cm⁻¹ and 1686 cm⁻¹ (attributed to conjugated C = O and C = C), along with alkane C–H stretches at 2965/2895 cm⁻¹ and a characteristic vinyl C–H out-of-plane band at 945 cm⁻¹. Accordingly, pyrethrin I and II were also expected to display broad C–H stretching vibrations in the (~2965–2895 cm⁻¹) range, a sharp C = O stretch around 1735 cm⁻¹, C = C stretching near 1686 cm⁻¹, and smaller CH bending bands (~1454, 1326 cm⁻¹). Cyclopropane and olefin signals typically appeared in the range of 970–945 cm < ge >. (Fig. 4A)

Fig. 4. Fourier-transform infrared spectroscopy (FTIR) of the ethanol extract: (A) A. pyrethrum; and (B) C. myrrha.

The FTIR spectrum of C. myrrha revealed characteristic peaks corresponding to the 12 major compounds (Fig. 7). A broad band observed around 3420 cm⁻¹ indicated O–H stretching, confirming the presence of alcohol groups. Peaks near 2970 and 2930 cm⁻¹ suggested N–H stretching, consistent with amine salts. The band at approximately 1740 cm⁻¹ was attributed to C = O stretching of esters, while the signal at ~1615 cm⁻¹ corresponded to C = C stretching, indicative of α, β -unsaturated ketones. Additional absorptions included ~1440 cm⁻¹ (O–H bending), ~1380 cm⁻¹ (S = O stretching), ~1245 cm⁻¹ (C–N stretching), ~1040 cm⁻¹ (S = O stretching), ~765 cm⁻¹ (C–Cl stretching), ~730 cm⁻¹ (C = C bending), and ~600 cm⁻¹ (C–I stretching), which were assigned to carboxylic acids, sulfonyl chlorides, amines, sulfoxides, halo compounds, and alkenes, respectively. (Fig. 4B)

Fig. 7. Root mean square deviation (RMSD) of the Nav1.7 protein over a 200 ns molecular dynamics simulation.

4. MTT cytotoxicity assay

Both A. pyrethrum and C. myrrha extracts were non-cytotoxic up to 250 µg/mL (IC₅₀) in the tested cell lines. This indicated high biocompatibility and safety, making these extracts suitable for inclusion in topical anesthetic gel formulations. (Fig. 5; Table 2).

Fig. 5. MTT cytotoxicity assay: A) Fibroblast cell line; B) Neural cell line. ^*P < 0.05 (one-way ANOVA).

Table 2. Percentage cell viability (average ± S.D.) and IC₅₀ values of extracts.

Cell Line	Sample	12.5 µg/mL	25 µg/mL	50 µg/mL	100 µg/mL	250 µg/mL	IC₅₀ (µg/mL)
Neural	A. pyrethrum	89.5 ± 4.2%	73.6 ± 3.8%	44.2 ± 2.5%	28.5 ± 1.9%	14.8 ± 1.2%	43.0 ± 0.25
	C. myrrha	98.1 ± 4.5%	77.4 ± 3.6%	63.1 ± 3.2%	38.4 ± 2.1%	25.2 ± 1.4%	54.0 ± 0.16
Fibroblast (L929)	A. pyrethrum	88.2 ± 3.5%	76.1 ± 3.1%	52.0 ± 2.8%	40.5 ± 2.2%	22.1 ± 1.5%	53.0 ± 0.21
	C. myrrha	94.0 ± 3.8%	78.5 ± 3.4%	56.2 ± 2.6%	42.0 ± 2.4%	26.5 ± 1.7%	57.0 ± 0.18

A. pyrethrum, Anacyclus pyrethrum; C. myrrha, Commiphora myrrha; IC₅₀, Half maximal inhibitory concentration; S.D., standard deviation.

5. In-silico docking studies

Molecular docking analysis revealed that both A. pyrethrin (Fig. 6A) and C. myrrha (Fig. 6B) exhibited significant binding affinities for all nine receptors, indicating strong interactions with ion channels and receptor complexes involved in neuronal signalling (Table 3). Nav1.7, a key voltage-gated sodium channel involved in pain transmission through the trigeminal nerve branches, is an important target for local oral anesthesia. This suggests their potential role in modulating ion channels contributes to their anesthetic effect.

Fig. 6. Molecular docking of: A) Pyrethrin; B) Furanoeudesma-1,3-diene; and C) Co-docking with 5EK0.

Table 3. Binding affinity values (kcal/mol) of Pyrethrin and Furanoeudesma-1,3-diene against selected neuronal ion channels and receptor proteins.

Receptor protein	Pyrethrin binding affinity (kcal/mol)	Furanoeudesma-1,3-diene binding affinity (kcal/mol)
NavAb voltage-gated sodium channel (6MVX)	-7.3	-7.4
Nav1.7 VSD2-NavAb chimera (6N4Q)	-6.1	-7.9
Human Nav1.7-VSD4-NavAb complex (5EK0)	-7.8	-7.9
Sodium channel protein type 4 (6AGF)	-7.1	-8.0
Chimeric human alpha1 GABA-A receptor (6CDU)	-6.8	-7.1
Apo chimeric GABA-A receptor (6D1S)	-8.2	-7.3
Potassium channel subfamily member (3UKM)	-6.5 to -7.6	-6.5 to -7.6
Potassium channel subfamily member (6RV3)	-6.5 to -7.6	-6.5 to -7.6
Sodium channel protein type 10 subunit alpha (7WE4)	-8.1	-7.5

MD simulations demonstrated stable binding of the phytoconstituents with the Nav1.7 channel over 200 ns. The Nav1.7–pyrethrin complex showed early equilibration with consistently lower RMSD values, indicating greater structural stability than the Nav1.7–myrrha complex, which exhibited increased deviation at later time points (Fig. 7). RMSF analysis revealed reduced residue-wise fluctuations in the pyrethrin-bound complex, suggesting restricted protein flexibility upon ligand binding (Fig. 8). Radius of gyration (Fig. 9) and SASA (Fig. 10) profiles further supported enhanced compactness and conformational stability of Nav1.7 with pyrethrin. Additionally, sustained higher hydrogen bond occupancy in the pyrethrin complex indicated stronger and more persistent ligand-protein interactions (Fig. 11). Collectively, these findings suggest pyrethrin exhibits more stable and favorable interaction with Nav1.7, supporting its potential role in modulating sodium channel activity during dental anesthesia.

Fig. 8. Comparison of residue-wise RMSF for the Nav1.7 protein-ligand complex over a 200 ns MD simulation. MD, molecular dynamics; ns, nanoseconds; RMSF, root-mean-square fluctuation.

Fig. 9. Radius of gyration of the protein over time, showing the compactness of the structure during the 200 ns MD simulation. MD, molecular dynamics; ns, nanoseconds; ps, picoseconds; Rg, radius of gyration.

Fig. 10. Ligand-induced modulation of solvent accessible surface area in Nav1.7.

Fig. 11. Number of hydrogen bonds between the protein and ligand over 200 ns simulation time, where Pyrethrin (blue) is compared with the standard drug-Myrrha (red).

Co-docking analysis demonstrated that both compounds simultaneously occupied receptor binding pockets without steric clashes, maintaining binding affinities comparable to individual results. Across sodium, potassium, and GABAA receptors targets, A. pyrethrin contributed hydrophobic and π-stacking interactions, while C. myrrha reinforced stability through hydrophobic interactions and occasional hydrogen bonding. The receptor ability to accommodate both ligands concurrently highlights a potential cooperative binding mechanism, suggesting molecular co-occupancy of ion channels or receptor function. These findings indicate promising prospects for multiligand approaches in drug discovery, particularly for targeting neuronal signalling pathways (Fig. 6C). A summary of the molecular and co-docking profiles is presented in Table 4.

Table 4. Molecular docking and co-docking interaction profiler summary.

Target receptor	PDB ID	Ligand	Binding affinity (kcal/mol)	Key hydrophobic interacting residues	Hydrogen bonds / π-stacking	Co-docking observation
NavAb sodium channel	6MVX	Pyrethrin	-7.3	Val1120, Leu1123, Ile1127, Ile1134, Leu2136, Phe2203, Val2204, Phe2207	π-Stacking: Phe2207	Simultaneous occupancy achieved without steric clashes. Affinities maintained (~ -7.3 to -7.4).
		Myrrha	-7.4	Phe2079, Phe2107, Val2110, Val2120	None	-
Nav1.7 VSD2-NavAb	6N4Q	Pyrethrin	-6.1	Leu875, Phe876, Tyr917, Val920, Phe921	None	Simultaneous occupancy confirmed. Myrrha maintains π-stacking.
		Myrrha	-7.9	Phe865, Phe891, Phe895, Ile923, Phe927	π-Stacking: Phe891	-
Nav1.7-VSD4-Na vAb	5EK0	Pyrethrin	-7.8	Met1640, Phe1644, Phe1670, Tyr1671, Trp1698, Ile1702	None	Cooperative binding suggested; both ligands stable in pocket.
		Myrrha	-7.9	Phe1644, Phe1670, Tyr1671, Phe1674, Trp1698, Ile1702, Phe1706	None	-
Nav1.4 sodium channel	6AGF	Pyrethrin	-7.1	Leu443, Phe797, Leu801, Ile1291, Val1590, Tyr1593, Ile1597, Leu1598	None	Simultaneous occupancy without structural hindrance.
		Myrrha	-8.0	Trp642, Phe645, Leu674, Phe677, Ile690, Phe1170, Phe1174, Ile1277, Phe1281	None	-
GABA-A receptor (Complex)	6CDU	Pyrethrin	-6.8	Ile239, Val243, Trp246, Thr306, Tyr309, Pro401	None	Active site accommodates both ligands. Myrrha H-bond preserved.
		Myrrha	-7.1	Leu56, Val58, Gln62, Ile67	H-bond: Tyr135	-
GABA-A receptor (Apo)	6D1S	Pyrethrin	-8.2	Pro51, Leu56, Glu59, Gln62, Ile67	H-bonds: Thr61, Gln62, Trp66	Strongest combined stability observed; high cooperative potential.
		Myrrha	-7.3	Ile239, Trp246	π-Stacking: Trp246	-
K1 potassium channel	3UKM	Pyrethrin	-7.2	Val139, Ile142, Pro143, Val192, Phe196, Phe220, Met260, Val263, Leu264	None	Simultaneous binding without steric clashes.
		Myrrha	-7.6	Leu147, Thr150, Leu273, Phe276	None	-
K3 potassium channel	6RV3	Pyrethrin	-6.5	Leu11, Leu115, Leu116, Pro119, Val123, Phe164, Phe238, Val242, Phe246, Phe1710	None	Both ligands share pocket maintaining distinct hydrophobic contacts.
		Myrrha	-6.9	Phe16, Leu20, Phe80, Phe84, Phe225	None	-
Nav1.10 sodium channel	7WE4	Pyrethrin	-8.1	Leu1285, Leu1289, Trp1292, Thr1365, Phe1710	π-Stacking: Phe1285	High binding affinity maintained in co-docked state.
		Myrrha	-7.5	Phe382, Phe386, Leu1614, Phe1655, Met1713	None	-

DISCUSSION

Herbs have historically played a significant role in anesthesia. This study aimed to develop a herbal alternative to topical anesthetic agents using A. pyrethrum roots and C. myrrha resin. In this study, ethanolic extracts of A. pyrethrum roots were used for phytochemical analysis, as ethanol is the most used solvent for extracting alkaloids with aesthetic properties [27]. While hexane is typically effective for extracting sesquiterpenoids from C. myrrha [20], this study showed that ethanol also yields substantial sesquiterpenoids.

HPLC is a sophisticated technique used for separating, identifying, and quantifying compounds in complex mixtures; offering high sensitivity, resolution, and reproducibility for phytochemical profiling. Patel VK et Al. established that the local anesthetic properties of A. pyrethrum extract primarily stem from its active alkaloid constituent, pyrethrin, which produces pyrethric acid [28].

GC-MS is another powerful analytical method for separating and identifying plant compounds. Phytochemical analysis of the ethanolic A. pyrethrum extracts confirmed pyrethrin alkaloids. This study also identified furanoeudesma-1,3-diene, which contributes to C. myrrha anethetic effect. This finding align with Ashrey El et al., who reported that Furanoeudesma-1,3-diene interacts with pain receptors [29], and exhibits local anesthetic activity by blocking inward sodium currents in excitable membranes. These findings confirm bioactive anesthetic compounds in both plant extracts, supporting their suitability for topical gel formulations.

The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay is a widely used colorimetric test for assessing compound cytotoxicity. To develop a topical anesthetic gel, ensuring the plant extracts are nontoxic to skin and fibroblast cells is essential for confirming biocompatibility. In this study, cytotoxicity effects of the ethanolic A. pyrethrum roots extract showed an IC₅₀ value of 53 ± 0.21µg/mL for neural cell lines. Bahri H et al. (2019) reported an IC50 value of 34.1µg/mL in a mouse neuroblastoma cell line [30]. Su S et al. (2011) assessed ethanolic C. myrrha extract against gynecological cancer cells, reporting an IC₅₀ of 26.63µg/mL [31]. In this study, cytotoxicity was minimal, with half of the cells remaining viable at 54µg/ml. Although this suggests a favorable safety profile in vitro, the relationship between these concentrations and actual clinical dose retained in the oral mucosa after gel application remains conjectural. Future permeation studies are necessary to determine whether active phytochemicals reach therapeutic threshold without exceeding cytotoxic limits in deep mucosal layers.

In silico molecular docking pyrethrin is relatively limited. According to Zhorov BS et al. (2016), docking simulations demonstrated that pyrethrin binds in a state-dependent manner, stabilizing the activated open channel state, resulting in continuous depolarization and paralysis in insects [32]. In this study, pyrethrin consistently exhibited strong binding affinities for all nine receptors, indicating potential as a potent modulator of neuronal ion channels and receptor complexes. This study provides the first molecular docking evidence supporting the potential anesthetic properties of furanoeudesma-1,3-diene through its interactions with neuronal channels.

To our knowledge, this is the first study combining A. pyrethrum and C. myrrha to evaluate their potential anesthetic properties. Co-docking results revealed that both pyrethrin and furanoeudesma-1,3-diene could simultaneously occupy neuronal ion binding channels, characterized by molecular co-occupancy. True pharmacodynamic synergy requires further quantitative validation using functional assays to differentiate between additive and cooperative effects.

Based on established pharmacological models for topical oral delivery, a 5% gel loading potentially yields a projected localized tissue concentration of 50–375 µg/mL [33]. This theoretical range aligns with the low micromolar binding affinities identified in our docking simulations; for instance, 50 µg/mL of the bioactive marker pellitorine (~224 µM) provides a substantial margin above predicted dissociation constants (Ki) required for Na_v1.7 and Na_v1.8 interaction [34]. While these estimated levels overlap with in vitro IC₅₀ values observed in this study (43.0–57.0 µg/mL), the anatomical complexity of the multi-layered oral mucosa and continuous salivary clearance may offer a significant physiological buffer [35]. This suggests the formulation can achieve receptor-level potency within a manageable safety window; however, these projections warrant further validation through in vivo permeation studies.

Although this study showed promising results, it has several limitations. Our results do not directly translate to clinical research. While docking scores indicate high binding plausibility, they do not provide functional evidence for sodium channel blockade or nerve conduction arrest. Additionally, determined IC₅₀ values suggest biocompatibility but remain clinically conjectural without permeation studies assessing actual dose retained at the oral mucosal site. Factors, such as plant sources, harvest conditions, and extraction yields can influence active marker concentration, potentially affecting biological effect reproducibility. Functional nerve conduction arrest is currently being validated through in vivo animal studies.

In conclusion, this study provides a comprehensive phytochemical and computational characterization of A. pyrethrum and C. myrrha as potential polyherbal alternatives for topical dental anesthesia. Analytical profiling confirmed key bioactive compounds, notably pyrethrin and furanoeudesma-1,3-diene, and cytotoxicity assays, indicated favorable biocompatibility in neural and fibroblast cell lines. Molecular docking and co-docking simulations suggested these compounds may interact with neuronal ion channels, specifically sodium and GABA-A receptors, through a plausible molecular co-occupancy mechanism.